U.S. patent application number 12/833510 was filed with the patent office on 2011-01-13 for antimicrobial composition and methods and apparatus for use thereof.
This patent application is currently assigned to FLORIDA GULF COAST UNIVERSITY. Invention is credited to Jose Barreto.
Application Number | 20110008469 12/833510 |
Document ID | / |
Family ID | 43427660 |
Filed Date | 2011-01-13 |
United States Patent
Application |
20110008469 |
Kind Code |
A1 |
Barreto; Jose |
January 13, 2011 |
ANTIMICROBIAL COMPOSITION AND METHODS AND APPARATUS FOR USE
THEREOF
Abstract
There is presented an alkaline disinfectant in the form of a
multi-component composition, with each component being relatively
benign until mixed with the other components in a ready-to-use
solution. The invention further relates to the use of the
composition in a variety of applications and apparatus for mixing
and dispensing thereof. Furthermore, the invention relates to the
use of components that synergistically provide effective biocidal
activity in a broad spectrum of organisms, including germs, molds,
viruses, bacteria, bacteria spores, or other microbes or pathogens.
The composition provides a chemical system that kills all known
plants, animals and microbes by raising pH and rapidly, but
indirectly, transporting hydroxide into cells by use of ammonia
compounds and/or amine compounds as neutral transporters. The
composition components synergistically operate together to provide
a disinfecting/cleaning composition useful for many different
applications.
Inventors: |
Barreto; Jose; (North Fort
Myers, FL) |
Correspondence
Address: |
HAHN LOESER & PARKS, LLP
One GOJO Plaza, Suite 300
AKRON
OH
44311-1076
US
|
Assignee: |
FLORIDA GULF COAST
UNIVERSITY
Fort Myers
FL
|
Family ID: |
43427660 |
Appl. No.: |
12/833510 |
Filed: |
July 9, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61224308 |
Jul 9, 2009 |
|
|
|
Current U.S.
Class: |
424/719 ;
514/578; 514/642; 514/663; 514/724 |
Current CPC
Class: |
A01N 33/04 20130101;
A01N 33/04 20130101; A01N 31/02 20130101; A01N 33/06 20130101; A01N
31/04 20130101; A01N 31/04 20130101; A01N 33/06 20130101; A01N
31/02 20130101; A01N 33/04 20130101; A01N 33/06 20130101; A01N
31/04 20130101; A01N 33/04 20130101; A01N 41/02 20130101; A01N
2300/00 20130101; A01N 25/30 20130101; A01N 2300/00 20130101; A01N
25/30 20130101; A01N 41/02 20130101; A01N 33/06 20130101; A01N
33/12 20130101; A01N 41/02 20130101; A01N 41/02 20130101; A01N
2300/00 20130101; A01N 59/00 20130101; A01N 59/00 20130101; A01N
2300/00 20130101; A01N 31/02 20130101; A01N 33/06 20130101; A01N
25/30 20130101; A01N 25/30 20130101; A01N 33/12 20130101; A01N
33/04 20130101; A01N 33/12 20130101; A01N 33/12 20130101 |
Class at
Publication: |
424/719 ;
514/724; 514/642; 514/578; 514/663 |
International
Class: |
A01N 31/00 20060101
A01N031/00; A01N 59/00 20060101 A01N059/00; A01N 33/12 20060101
A01N033/12; A01N 41/04 20060101 A01N041/04; A01N 33/02 20060101
A01N033/02; A01P 1/00 20060101 A01P001/00 |
Goverment Interests
GRANT REFERENCE
[0002] The research carried out in connection with this invention
was supported in part by a grant from the Department of Defense and
the Environmental Protection Agency [EM-83298201-1]. The Government
has certain rights in the invention.
Claims
1. A biocidal composition comprising: an amount of at least one
first component designed to suppress ionization and generate a
permeable hydroxophore in a solution of components in the
composition, an amount of at least one second component designed to
accelerate the net transfer of hydroxide ions across the organism
plasma membrane of the microbe or pathogen, an amount of at least
one third component designed to disrupt cell membranes and invert a
membrane potential, wherein at least one of the components causes
fluidization of a cell membrane of a microbe or pathogen, wherein
the at least three components, when combined with an external
alkaline pH gradient, generate an alkaline flux which leads to cell
death by rapidly raising the pH of the cellular cytoplasm.
2. The composition of claim 1, wherein the killing mechanisms of
the composition are selectively switched off by neutralization.
3. The composition of claim 1, wherein the composition leaves
little to no residual toxicity upon neutralization of the
composition.
4. The composition of claim 3, wherein neutralization occurs by
evaporation of one of more of the components or by the application
of a neutralizing agent.
5. The composition of claim 1, wherein the at least one first
component is a long chain detergent material in a concentration of
mM range.
6. The composition of claim 1, wherein the at least one second
component is a hydroxophore material in a concentration of mM
range.
7. The composition of claim 1, wherein the at least one third
component is an alcohol in a concentration of mM range.
8. The composition of claim 1, wherein the concentration of the at
least one first component is in the range of about 5-40 weight
percent relative to the at least three components in the
composition, the at least one second component is in the range of
about 5-40 weight percent relative to the at least three components
in the composition, and the at least one third component is in the
range of about 1-10 weight percent relative to the at least three
components in the composition.
9. A process for preparing a biocidal composition, the process
comprising: providing an amount of at least one first component
designed to suppress ionization of components in the composition,
an amount of at least one second component designed to create an
alkaline environment of non-ionized constituents around the cell
membrane of the microbe or pathogen, and an amount of at least one
third component designed to reverse the cell membrane potential,
with each of the components stored separately, and mixing of the
components for deployment to decontaminate surfaces or areas.
10. The process of claim 9, wherein the at least one first
component is a long chain detergent material in a concentration of
mM range.
11. The process of claim 10, wherein the at least the at least one
first component is a long chain detergent.
12. The process of claim 9, wherein the at least one second
component is a hydroxophore material in a concentration of mM
range.
13. The process of claim 9, wherein the at least one third
component is an alcohol in a concentration of mM range.
14. A biocidal composition comprising: at least an amount of a
first component being a detergent material in a concentration of mM
range, at least an amount of a second component being a
hydroxophore material in a concentration of mM range, at least an
amount of a third component being an alcohol in a concentration of
mM range, wherein the at least three components, when combined with
an external alkaline pH gradient, act as a biocidal agent.
15. The composition of claim 14, wherein the first component is a
long chain detergent material.
16. The composition of claim 14, wherein the killing mechanisms of
the composition are selectively switched off by neutralization.
17. The composition of claim 16, wherein neutralization occurs by
evaporation of one of more of the components or by the application
of a neutralizing agent.
18. The composition of claim 16, wherein the composition leaves
little to no residual toxicity upon neutralization of the
composition.
19. The composition of claim 14, wherein the detergent material is
selected from the group consisting of ammonia, cetyl
trimetylammonium chloride, cetyl trimethylammonium bromide,
tetradecyl trimethyl ammonium chloride, tetradecyl trimethyl
ammonium bromide, sodium dodecyl sulfate, sodium tetradecyl
sulfate, sodium cetyl sulfate, sodium octyl sulfate,
hexadecylamine, tetradecylamine,
3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate,
3-(decyldimethyl-ammonio) propane sulfonate or combinations
thereof.
20. The composition of claim 14, wherein the alcohol is selected
from the group consisting of methanol, ethanol, propanol, butanol,
benzyl alcohol or combinations thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims the benefit of U.S.
Provisional Patent Application No. 61/224,308 filed Jul. 9, 2009,
the disclosure of which is expressly incorporated by reference
herein.
FIELD OF THE INVENTION
[0003] The invention relates to antimicrobial compositions and
methods and apparatus for effective use thereof. More particularly,
the invention relates to antimicrobial compositions useful for a
variety of purposes in cleaning and disinfection, and methods and
apparatus for using and dispensing the compositions.
BACKGROUND OF THE INVENTION
[0004] Antimicrobials are generally used to destroy or suppress the
growth or reproduction of microbes such as bacteria, viruses and
the like. Many commonly available germicides are too toxic,
excessively persistent, or difficult to handle in certain
applications. For example, germicides using ozone, bleach, ethylene
oxide, and various other oxidizing or halogenating chemicals fit in
this category. Such germicides use a harsh chemical toxicity to
engender killing but, whenever biocides are used, a balance must be
struck between reducing an infectious microbial hazard and the
creation of a serious chemical hazard. Antimicrobial compounds may
act on targeted microbes in a variety of ways. For example, the
antimicrobial compound may alter the cell wall of a microbe, either
by altering cell wall permeability or by altering cell wall
synthesis and repair, to destroy the microbe. Other compounds may
prevent DNA or protein synthesis to destroy the microbe. While
there are numerous known antimicrobial compounds, and numerous
known mechanisms by which antimicrobial compounds may function,
such compounds have various deficiencies. There are many
characteristics that can be relevant when trying to decide whether
or not a particular compound is useful as an antimicrobial.
Relevant factors may include the relative potency of the compound
against a specific microbe or against a spectrum of microbes, and
the relative selectivity of the antimicrobial activity of the
compound in targeting microbes or pathogens. Other factors may
relate to the compounds producing undue irritation to eyes or skin
at levels required to impart germicidal properties. Some
antimicrobial compounds can be dangerous to use at higher
concentration levels, or may leave residues that can continue to
cause physical implications in humans. There are also long-term
concerns, including the likelihood that the microbe may develop
resistance to the antimicrobial compound. Additionally, additional
concerns may relate to the cost and commercial availability of the
antimicrobial compound.
[0005] Disinfecting compositions are commercially important
products and enjoy a wide field of utility in assisting in the
disinfecting and cleaning of surfaces, such as for use in cleaning
"hard surfaces". Hard surfaces are countertops, walls, floors and
other such surfaces, such as in the home in kitchens, bathrooms,
etc, and in hospitals or the like, where cleaning and disinfecting
of such surfaces is important. Various formulations of
cleaning/disinfecting agents have been developed, but may not be as
effective at disinfecting and cleaning as desired. There is thus a
current and continuing need for cleaning/disinfecting products
which are highly effective disinfectants, but leave a minimum of
discernible residue and are easily used and produced
inexpensively.
[0006] In some applications, the materials used in cleaning or
purifying materials may have other deficiencies. In water treatment
for example, compounds such as chlorine are commonly used, but are
toxic, and therefore may cause potential problems. For example,
chlorination for the disinfection of raw water may produce
trihalomethanes (THM's), such as chloroform, which are
carcinogenic. It has further been determined that chlorinated
drinking water, when ingested by laboratory animals, has also shown
signs of carcinogenic effects. Handling of the material is also
hazardous, putting those working with it at risk. Other water
treatment approaches are costly or less effective than desired.
[0007] Currently, there are also heightened concerns over the
hazards produced by microbes and pathogens, including in relation
to the potential of bioweapons engineering of microbes or pathogens
that could be released to impact a population. Other concerns
relate to the possible release of biohazardous materials by
accident. Government entities have therefore begun steps of
preparedness for handling such incidents should they arise.
Sterilization after a biological weapon attack is problematic when
using overly harsh sterilizing agents. Although such germicides
will destroy pathogens, they also damage the environment, and can
result in damage to sensitive electronic equipment, furnishings and
documents, can corrode metals, and harm human health. As a
consequence, most germicides have a narrow scope of use and/or
require extensive training for safe deployment.
[0008] Accordingly, new antimicrobials and germicides, and new
sources of antimicrobials are desired and are increasingly
valuable. It would also be highly desirable to provide
antimicrobial compositions that achieve the characteristics of
providing broad spectrum antimicrobial activity, while minimizing
eye or skin irritation or other harmful effects, and while
providing desired cleaning efficacy.
SUMMARY OF THE INVENTION
[0009] In general, this invention relates to an alkaline
disinfectant in the form of a multi-component composition, with
each component being relatively benign until mixed with the other
components in a ready-to-use solution. The invention further
relates to the use of the composition in a variety of applications
and apparatus for mixing and dispensing thereof. Furthermore, the
invention relates to the use of components that synergistically
provide effective biocidal activity in a broad spectrum of
organisms, including germs, molds, viruses, bacteria, bacteria
spores, or other microbes or pathogens. The composition provides a
chemical system that kills all known plants, animals and microbes
by raising pH and rapidly, but indirectly, transporting hydroxide
into cells by use of ammonia compounds and/or amine compounds as
neutral transporters. The composition components synergistically
operate together to provide a disinfecting/cleaning composition
useful for many different applications. In an example, the
composition comprises 1) ethanol, 2) cetylamine, 3) ammonia and 4)
a pH greater than 9.
[0010] The germicide according to the invention has the following
characteristics. Extreme lethality, with no pathogens remaining to
cause infection. A broad spectrum of killing activity is provided,
with rapid killing of pathogens. The germicide has a short
persistence time, with no long term toxicity remaining, and also
has "switchability, wherein chemical neutralization may be used as
a switch to immediately deactivate the germicide. Further, as
components have high vapor pressures, evaporation of several
components after deployment of the biocide provides an
"auto-switch", so that after sterilization, no long term toxicity
can occur. The germicide also is inexpensive, portable, storable,
safe and easy to use. The germicide and methods and apparatus for
dispensing provide a system, wherein multiple components are safely
provided in a storable arrangement, and easily mixed and used to
provide for extreme lethality of microbes and pathogens. In an
example, a dispensing device design allows the components to be
stored safely for long periods in a multi-compartment plastic
cylinder. When use is desired, mechanical rupture of plastic
membranes is followed by mixing to obtain the germicidal
composition and activity. A method for the cleaning of hard
surfaces comprises the steps of providing the components for
preparing a germicidal composition as described above; selectively
mixing the components just before deployment, diluting the mixed
composition with up to about 500 parts by weight water; and
contacting the diluted cleaning composition with a hard surface to
thereby kill microbes or pathogens that may be on the surface.
[0011] It is therefore an object of the invention to provide a
germicidal disinfectant which conforms to these requirements, which
can be formulated as a storable concentrate, and can be selectively
diluted to form an aqueous, ready-to-use solution. The invention
includes both methods and compositions to achieve the desired
results, as described, but is not limited to the various
embodiments disclosed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a graph representing the effects of ethanol in
differing concentrations on test bed organisms;
[0013] FIG. 2 is a graph representing the effects of hexadecylamine
in differing concentrations on test bed organisms;
[0014] FIG. 3 is a graph representing the effects of ammonia in
differing concentrations on test bed organisms;
[0015] FIG. 4 is a graph representing the effects of differing pH
on test bed organisms; and
[0016] FIG. 5 is a graph representing the killing efficacy of the
composition according to an example of the invention and other
killing agents.
[0017] FIG. 6 is a graph representing the killing efficacy of a
composition according to an example of the invention with varying
detergent materials.
[0018] FIG. 7 is a graph representing the killing efficacy of a
composition according to an example of the invention with varying
alcohol materials.
DETAILED DESCRIPTION OF THE INVENTION
[0019] Turning to a first example according to the invention, the
composition of the germicide comprises a synergistic combination of
components that together utilize an inward flux of chemicals that
immediately interferes with cell metabolism and viability. The
composition uses multiple components and multiple mechanisms to
achieve complete lethality of microbes and pathogens of various
types, such as including germs, molds, viruses, bacteria, bacteria
spores, or other microbes or pathogens. The composition generates
chemical flux into such microbes and pathogens that is invariably
lethal to the microbes and pathogens. Although there may be
extremophile organisms, occupying certain rare environments on
earth, are capable of resisting the killing mechanism engendered by
the composition of the invention, these are not human pathogens.
Further, by using longer, or more concentrated treatment, even
these extremophiles can be effectively killed by the germicide.
[0020] In general, the germicidal composition of the invention is
alkaline, and has at least three components which act
synergistically to kill germs, microbes and pathogens, and the
mechanism of action is generic and can kill various microbes and
pathogens including bacteria, bacterial spores, molds, and viruses
for example. As with other components as will be described, each is
in very small concentrations in the mM range. The first component
is a small amount of ammonia or another long chain detergent type
material for example, in the mM range, and in an amount of between
5-40 weight percent for example relative to the other components.
Other concentrations may be suitable depending on the application.
In this example, ammonia can exist in two forms in water, free base
ammonia (a gas solvated in liquid water; NH.sub.3) and a protonated
form, the ammonium ion (NH.sub.4.sup.+). The free base ammonia
(NH.sub.3) is readily permeable through the plasma membrane of all
living cells and the free base form is more prevalent above the pKa
of 9.0. That is to say, above a pH of .about.9.0 the free base will
predominate. If the external pH of a cell is raised above 9.0 (and
maintained at that pH) the cell will eventually die because an
alkaline flux will ensue, either hydroxide ions will enter the cell
or hydronium ions will leave, these ion fluxes will occur
spontaneously because pH gradients are not stable, and will
collapse to the lowest energy level, meaning that a pH gradient
across a semipermeable membrane will eventually collapse to yield
the same pH on the inside and the outside of a membrane separating
two compartments. In the example above, the pH will eventually
become 9.0 on both sides of the compartment, and the biocide of the
invention is designed to increase the rate of pH gradient collapse.
A cell with a cytoplasmic pH of nine will die, because enzymes and
proton gradients within the cell are function at a neutral pH of
about 7.0 for most living organisms. Prolonged or severe
alkalinization kills all known living cells. Even alkaline
extremophiles deal with an alkaline external pH by using hydroxide
pumping mechanisms, and even they cannot tolerate an alkaline
internal pH, with the biocide killing even alkaline extremophiles
by overwhelming the pumping mechanisms. In the germicide of the
invention, the ammonia or other long chain detergent compound
functions as a hydroxophore, and free base ammonia enters a cell in
the neutral form and accepts a proton. The loss of a proton inside
the cell is an event that corresponds exactly to the entry of a
hydroxide anion because at a given pH, hydroxide anions and protons
are linked by an equilibrium constant, it is impossible to change
the concentration of hydroxide without altering the concentration
of protons. In summary, the ammonia or other long chain detergent
component results in accelerating the rate of alkalinization. A
variety of other base materials to increase alkalinization may also
be suitable.
[0021] Further examples of detergents that have been shown to be
effective for use in the composition and method of the invention
are set forth in Table 1 below, but it is understood that this list
is not limited, and other detergents or agents providing the
function of the detergent component may also be suitable.
TABLE-US-00001 TABLE 1 Number of Name Abbreviation Carbons Full
Name CTAC 16 Cetyl trimetylammonium chloride CTAB 16 Cetyl
trimethylammonium bromide TTAC 14 Tetradecyl trimethyl ammonium
chloride TTAB 14 Tetradecyl trimethyl ammonium bromide SDS 12
Sodium dodecyl sulfate STS 14 Sodium tetradecyl sulfate SCS 16
Sodium cetyl sulfate SOS 8 Sodium octyl sulfate HAD 16
Hexadecylamine TDA 14 Tetradecylamine CHAPS XX 3-[(3-
Cholamidopropyl)dimethylammonio]- 1-propanesulfonate DAPS XX
3-(decyldimethyl-ammonio) propane sulfonate
[0022] A second component, being a second hydroxophore is present
in the biocide composition, such as hexadecylamine (HDA) or aniline
as particular examples. The concentration of this component may be
in the range of 5-40 weight percent for example, relative to the
other components. Other concentrations may be suitable depending on
the application, and as with the first component, again is
generally in the mM range in a concentrate formulation that is
dilutable for use. The HDA molecule for example has a long
hydrophobic tail and an amine head group. The hydrophobic tail
initially inserts into the plasma membrane of living cells and the
amino head group remains in the water exposed to the external
compartment. Compounds like HDA are known to flip-flop in the
membrane so that the free amino group can travel from the external
compartment to the internal compartment, but the molecule remains
anchored in the plasma membrane. When flip-flop does occur, a
hydroxophore action is evident, since a free ammonia group has just
been transported from the external to the internal compartment.
Note that this action is synergistic with the first component, such
as free ammonia. A second damaging event conferred by HDA has to do
with disruption of the plasma membrane caused by the insertion of
the hydrophobic tail, HDA is essentially a detergent and will cause
increased fluidity and disruption of the membrane, with such
disruption leading to increased permeability of ammonia and an
increase in flip-flop. Lastly once the HDA head group appears in
the internal compartment it becomes protonated turning into
ammonium. Effectively, this creates increased positive charge on
the inner face of the membrane. Most plasma membranes in living
cells maintain an external positive charge, HDA or other suitable
materials therefore changes the membrane potential creating a
positive charge on the inner leaflet of the phospholipids in the
plasma membrane. All three actions of HDA are deleterious to living
cells and synergistic with the first component such as ammonia.
Alternatively, this component may be substitution of aniline for
HAD, which also created a very lethal biocide and confirms a
mechanism of action.
[0023] A third component in the biocide composition is an alcohol,
such as ethanol. The concentration of this component may be in the
range of 1-10 weight percent for example, relative to the other
components. Other concentrations may be suitable depending on the
application. Ethanol freely dissolves in the hydrophobic core of
the plasma membrane and fluidizes that membrane. The fluidization
caused by ethanol is synergistic with the fluidization effects of
HDA. Ethanol also changes the polarity of water making the solvent
much less polar, this circumstance causes more free base ammonia to
appear since that decrease in polarity makes the ammonium ion much
less stable and favors an increase in the percentage of amine free
bases. Ethanol is therefore synergistic in causing the hydroxophore
action to increase, and helps cause a rapid increase in
alkalinization. Other alcohols in relatively low concentrations may
also be used. For example, alcohols such as including but not
limited to methanol, propanol, butanol, benzyl alcohol have been
found to be effective in the composition and method of the
invention.
[0024] The biocide composition of the invention greatly increases
the toxicity by increasing the rate of alkalinization and can kill
very quickly compared to other alkaline biocides. The biocide of
the invention will also "auto-switch" off when the ethanol and
ammonia evaporate, or it can manually be "switched off" by
neutralizing it with non-toxic acetic or citric acid. Due to the
synergistic effects of the components, the biocide results in rapid
killing of the organisms, which is very desirable. For example,
data indicate that the biocide achieves complete sterilization in a
few minutes using a test bed organism, Vibrio fischeri. For a
variety of applications, such as bio-terror attacks with pathogens,
the "switchable" biocide/germicide of the invention would provide
lethality to all known pathogens, while allowing the killing
mechanism to be switched off after sterilization. The biocide uses
alkalinity as the primary germicidal agent, and the lethality of
the biocide can thus be instantly switched off by neutralization.
At the same time, the individual components are individually
benign, so that the separate liquid components can be stored for
extended periods before use is desired, and pose no threat prior to
mixing in just prior to use or in the field. The components will
not significantly degrade upon long-term storage. Thus, the three
components of the biocide composition when combined with
alkalinity, these agents produce very effective biocides, but
before mixing, the three components alone are relatively benign and
pose little threat to humans, such as on skin contact. Also, due to
the small concentrations, even upon contact with mucous membranes
of the eyes, mouth or nose, though there may be slight tissue
injury because those cells are permeable to the agents, the effects
are minimized.
[0025] The proposed mechanism of action of the biocide includes a
membrane fluidizing agent, which also helps generate a permeable
hydroxophore in solution. A second agent disrupts cell membranes
and inverts a membrane potential, while the third agent is a
hydroxophore, and it accelerates the net transfer of hydroxide ions
across the organism plasma membrane. The three components, when
combined with an external alkaline pH gradient, generate a large
alkaline flux which leads to cell death by rapidly raising the pH
of the cellular cytoplasm. Notably, this killing mechanism can be
easily "switched" off by neutralization to leave little to no
residual toxicity after the biocide has served its purpose.
Example 1
[0026] V. fischeri was grown overnight until the culture reached
log phase and were brightly luminescent. Since bacterial
luminescence is an ATP driven event, light emission is directly
related to the metabolism and energy charge state of the organism.
Lower luminescence levels correlate to lowered metabolism levels
when compared to a non-treated control. It was previously
established that when treated with known biocides, luminescence is
abolished and does not recover. Samples at given concentrations
were added to V. fischeri in a 24 well plate. A small amount was
placed in a 96 well plate, agitated, and the luminescence was read
in a TECAN well plate reader. For all figures, the total time
period that V. fischeri was exposed to each biocide or component
was between five and fifteen minutes. Results of testing show that
Vibrio fischeri react to some of the individual components of the
alkaline biocide in different ways. As shown in FIG. 1, the effect
of ethanol at concentrations from 0%-10% on V. fischeri
Luminescence are shown. The left side bars represent final pH of 7.
The right side bars represent a final pH of 9. Low readings
indicate killing. As seen in FIG. 1, V. fischeri is susceptible to
ethanol; with even 1% concentrations show damage at pH 7 (and also
at pH 9). V. fischeri treated with samples at a final pH of 9
showed higher luminescence than those treated with samples at a
neutral pH. In other words, at a given concentration of ethanol, pH
7 seems to be slightly more damaging than the same concentration at
pH 9. For example V. fischeri treated with 3% ethanol at pH 9 had a
40% higher luminescence reading than the same concentration at pH
7. This could be a result of stimulating the luminescence pathway
through "irritation". Mildly alkaline environments may not be harsh
enough to damage the cell but may be irritating enough to enhance
the luminescence pathway. It is important to note that while a
luminescence of 0% of control is designated as "killed", a
luminescence higher than 100% of control is more difficult to
interpret and may be due to the fact that the bacteria can generate
a higher luminescence when stressed. From these test results,
ethanol is the most lethal component of the alkaline biocide of the
invention for V. fischeri. The purpose of this component is to
fluidize the cell membrane to collapse the pH gradient. For other
organisms, this component may be less effective than other of the
components, wherein the combination of components synergistically
provides for effective killing over a broad spectrum.
[0027] Turning to FIG. 2, the effect of the component HDA or a
cetyl amine ranging from 0 mM-2 mM on V. fischeri luminescence was
tested. The left side bars represent final pH of 7.5, while the
right side bars represent a final pH of 9. FIG. 2 shows that HDA
has little to no effect on V. fischeri at pH 7.5, however it does
have a slight effect at higher concentrations at pH 9. A 29%
decrease in luminescence is seen at 2 mM C2 at pH 9. At pH 7.5 2 mM
C2 only shows an 11% decrease in luminescence. This does not follow
the earlier trend of higher luminescence at higher pH. In the
biocide, the ethanol and ammonia components are volatile. The HDA
component has a very low solubility in water, but this is not a
drawback because it is effective at low concentration (in
combination with the ethanol and ammonia type components) and this
minimizes any residual residue that would be left behind after
using the biocide. The presence of ethanol or the like further
increases the solubility level of the HAD or the like. For some
applications, higher concentration of HDA or the like may be
suitable, and once the three components are mixed, the solubility
level of HDA will rise.
[0028] Turning to FIG. 3, the effect of ammonia ranging from 0
mM-50 mM on V. fischeri Luminescence is shown. The left side bars
represent final pH of 7, while the right side bars represent a
final pH of 9. FIG. 3 illustrates that the component ammonia by
itself shows no difference from the control (pH adjusted H.sub.2O)
at either pH 7 or 9, meaning V. fischeri is not affected by ammonia
at 50 mM concentration or lower at these pHs. This is an important
finding because the flux of OH-- into the cell is the major source
of sequestration of H+ ions, which causes the interior cell
cytoplasm to alkalinize and eventually leads to cell death. The
flux of OH-- into the cell is the major killing event induced by
this component. Samples with very high ammonia concentrations are
not killing the cell any faster than samples with little or no
ammonia. This means that without the other components, in the case
of V. fischeri, ammonia at these concentrations alone does not
produce enough of an alkaline flux to damage the cell. The
components work synergistically in order for the biocide to work
properly. Samples with very high C3 concentrations are not killing
the cell or sequestering H+ any faster than samples with little or
no C3. This means that without the other components, in the case of
V. fischeri, C3 alone does not produce enough of a hydrogen ion
flux to damage the cell. The other components are necessary in
order for the biocide to work properly.
[0029] Turning to FIG. 4, the effect of pH from 7-10 on V. fischeri
Luminescence is shown. An external pH gradient drives an influx of
OH.sup.- into the intracellular environment and concomitantly
creates a H.sup.+ gradient driving an H.sup.+ efflux, either
pathway collapses the pH gradient and will alkalinize the
cytoplasm, killing the bacterium. FIG. 4 shows that alkaline pH
alone does not kill V. fischeri. All samples tested showed an
unchanged or higher luminescence than the H.sub.2O control and the
untreated control. As noted previously, a higher luminescence is
seen with increased alkalinity (see FIG. 1). An extracellular
alkaline environment seems to increase the luminescence. Whether an
increase in cellular metabolism is producing a higher number of
luminescent molecules per cell, or there is simply a "mild
"irritant" effect, or the V. fischeri thrive at a higher pH is not
yet determined.
[0030] Turning to FIG. 5, the effect of various killing agents on
V. fischeri Luminescence are shown. Samples are shown at their
final concentrations. The biocide listed on the far left is the
biocide according to the invention, with a combination of the three
components as described above, at pH 9. As seen in FIG. 5, the
alkaline biocide according to the invention (listed as biocide in
FIG. 5) is completely lethal to V. fischeri along with the other
very harsh sterilizing agents shown in the figure. The advantages
of the multi-component biocide according to the invention, such as
compared with these other harsh sterilizing agents are as follows.
Extreme lethality is generated, with no pathogens remaining to
cause infection. The biocide of the invention utilizes an inward
flux of chemicals that immediately interferes with cell metabolism
and viability. The multiple components and multiple mechanisms
provided by the biocide of the invention achieve complete
lethality, with the chemical flux generated being invariably lethal
to V. fischeri. Accordingly, extrapolation indicates that the
biocide would be similarly lethal to any other microbes or
pathogens. The biocide thus has a broad spectrum of killing
activity is desired, with an overall mechanism that will destroy
any known (or newly created) pathogen. The biocide achieves rapid
killing, which is very desirable. The data indicate that complete
sterilization is achieved at least in about 5-15 minutes using the
test bed organism, Vibrio fischeri. The biocide has a short
persistence time, with no long term toxicity remaining. As
described, each component in the multi-component water based system
is relatively harmless but, when combined and diluted, produces a
potent biocide. The biocide is switchable between being a highly
effective killing agent, while allowing instant chemical
neutralization as a switch to immediately deactivate the
biocide/germicide. Further, as several of the components have high
vapor pressures, evaporation of these components after deployment
of the biocide provides an "auto-switch", so that after
sterilization, no long term toxicity can occur. The germicide also
is inexpensive, portable, storable, safe and easy to use. The final
concentration of the components is small and therefore, the
composition is less harsh on the surfaces or materials it is
used.
[0031] Turning to FIGS. 6 and 7, there is illustrated the principle
that a wide variety of detergents or alcohols work as a component
of the biocidal composition. In the first experiments shown in FIG.
6, the concentration of ammonia was held constant at 1 mM, while
the ethanol concentration was held at 0.5% and the pH was adjusted
to 9. In this example, the final concentrations in the biocide are
1 mM NH3, 0.5% EtOH, and 0.05 mM of detergent, and pH was adjusted
to 9.0 with 1.0M sodium hydroxide. The type of detergent was then
varied at a concentration of 0.05 mM. The detergents used in these
examples were CTAC, CTAB, TTAC, TTAB and SDS. All of the detergents
showed some degree of killing, with it being understood that the
detergent concentration of 0.05 mM was very low. The use of very
low detergent concentration was purposefully done in these examples
in order to show variations in the killing power of the different
detergents. If the detergent concentration was raised to 1 mM
(which is still very dilute), all the detergents were found to kill
effectively. Similarly, analogous experiments were conducted to
show the killing power of various alcohols within a constant
formulation. In the experiments shown in FIG. 7, the composition
included an ammonia concentration that was held constant at 1 mM, a
CTAB concentration of 0.05 mM and the pH was adjusted to 9. In this
example, the final concentration in the biocide are 1 mM NH3, 0.05
mM CTAB, and 0.5% of alcohol, and pH was adjusted to 9.0 with 1.0M
sodium hydroxide. The type of alcohols were then varied at a
concentration of 0.5%. The alcohols used were methanol, ethanol,
propanol, butanol and benzyl alcohol. All of the alcohols showed
some degree of killing, with it being understood that the alcohol
concentration of 0.5% was very low. The low concentration of
alcohol was purposefully done in order to show variations in the
killing power of the alcohols. If the alcohol concentration was
raised to 1% (which is still very dilute), all the alcohols were
found to kill effectively.
[0032] In the composition, the particular components are only
examples, and other similar components are contemplated. The
components work together to greatly increase killing efficacy. The
components each have mechanisms, that work synergistically together
to increase the biocidal activity. For example, ethanol or the like
suppresses ionization, such that the ammonia tends to exist in
neutral form that allows the ammonia (or the like) to enter the
cell very rapidly when neutral ammonia (or the like) builds up
around the pathogen. The component of ethanol, or the like,
suppresses ammonium ion creation and creates more free ammonia (or
the like). Further, the ethanol or the like fluidizes the cell
membrane to increase the flux of ammonia into the microbes or
pathogens. The HDA or the like component also assists by reversing
the membrane potential, and fluidizes the membrane and carry a free
ammonia into the cell and take a positive charge from the outside
of the membrane to the inside. Flipping the electrical potential of
the membrane also causes problems in cell function to facilitate
killing efficacy.
[0033] Further, the biocide composition chemicals involved are very
inexpensive, and the components are stable and do not decompose
during long term storage. A high initial concentration of each
component enables portability when water is available. Mixing the
components immediately creates the potent germicide. Spraying and
soaking are acceptable modes of application. Thus, the composition
may be stored as the individual components in a suitable container
or storage system, until use of the biocide/germicide is desired,
and then mixed and deployed as needed. As merely examples, the
components may be stored in a container and separated by a
frangible material until use is desired, wherein the frangible
material can then be ruptured to cause mixing of the components for
use. Alternatively, the components could be stored separately and
combined and mixed at a spray nozzle for example. Any other
suitable arrangement for selectively storing the components and
subsequently mixing them for use are contemplated. Further, the
components may be mixed and stored in a container for use if
desired, such as via a spray bottle or the like.
[0034] For example, an application of the biocide/germicide may
relate to cleaning and disinfecting of household surfaces, such as
kitchen counters, bathroom surfaces, walls, floors, etc., similar
to many household types of cleaning/disinfecting agents currently
in the market. Other areas, such as in hospitals, ambulances,
airplanes or a variety of other environments and applications are
also suitably decontaminated using the composition of the
invention. Cleaning of industrial food processing equipment or
other industrial machines may also be possible. The advantages of
the present invention allow for highly effective disinfection of
such hard or other surfaces, while not leaving any lasting toxicity
or unwanted residue, and not leaving any harsh chemicals that could
affect people adversely. The composition may be used in a manner
similar to other such cleaning/disinfecting products by mixing with
water and applying to surfaces via wiping or the like, or by
spraying or other suitable means.
[0035] The composition could also be used in the
disinfection/decontamination of water or other fluids, such as in a
water treatment facility. For example, water to be treated may be
contained in a tank or the like, and a suitable amount of the
composition of the invention may be introduced into the water.
Mixing of the composition with the body of water may allow
dispersion of the biocide to treat the totality of the water.
Mixing or bubbling of the water after some dwell time may also
allow the volatile components to evaporate more quickly to switch
off the disinfection activity and leave the water decontaminated.
Such a process could also be used in other applications such as
fish, shrimp or the like farms, where fish or shrimp are grown in a
pond or the like. In such situations, it is possible for the pond
to become contaminated with bacteria or viruses for example, that
can decimate the animal population. Further, in many such
environments, it is possible that water from other sources, such as
a nearby body of water or bay, comingles into the pond, either
potentially bringing in pathogens to the pond water, or allowing
escape of contaminated pond water into such other bodies of water.
Such problems not only cause significant damage to the farming
activity, but any such releases can also impact wildlife or
vegetation in the other bodies of water. The disinfecting
composition of the invention may be used to sterilize water coming
into and out of such ponds or the like. Other disinfectants, such
as chlorine cannot be used because they introduce halogenated
compounds into the environment. In an example, water supplied to or
from such a pond could be first sent to a holding pond to
clean/decontaminate the water coming in or to be released, with the
biocide composition introduced into the holding pond for treatment
of the water. Upon being decontaminated, the composition may then
be switched off and the decontaminated water introduced into or
released from the pond. As an example, the switching off of the
biocidal activity of the composition may be performed by air
bubbling in the holding tank which accelerates removal of the
volatile components by evaporation. Testing of the pH level of the
water in the holding tank will indicate when it is switched off,
such as at around a pH of 7. Other water treatment applications are
contemplated, such as treatment of ballast water from vessels for
example.
[0036] As another example, the biocidal composition may be used to
remediate bio-terror attacks with pathogens, or spills or other
releases of pathogens, the biocide of the invention may be deployed
over large areas for sterilizing the pathogens quickly, and then
the lethality of the biocide could be instantly switched off by
neutralization. The components may be stored in large containers
and individually pumped to a spray nozzle where they can be mixed
with water for deployment. Alternatively, the components could be
mixed in the field and deployed by spraying or soaking.
[0037] Based upon the foregoing disclosure, it should now be
apparent that the methods of producing biocidal/germicidal
compositions, methods and devices provide a highly effective system
for decontamination of surfaces, water or other fluids, and many
other applications that are too numerous to describe. The
components used in the composition can be modified while still
performing the synergistic functions with one another. Example
apparatus for the deployment of the decontamination composition are
likewise too numerous to describe, as a wide variety of devices are
envisioned. It is, therefore, to be understood that any variations
evident fall within the scope of the claimed invention and thus,
the selection of specific component elements can be determined
without departing from the spirit of the invention herein disclosed
and described.
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